Modulation of miR-145-5p and miR-146b-5p levels is linked to reduced parasite load in H9C2 Trypanosoma cruzi infected cardiomyoblasts

In the heart tissue of acutely Trypanosoma cruzi-infected mice miR-145-5p and miR-146b-5p are, respectively, downregulated and upregulated. Here, we used the H9C2 rat cardiomyoblast cell line infected with the Colombian T. cruzi strain to investigate the parasite-host cell interplay, focusing on the regulation of miR-145-5p and miR-146b-5p expression. Next, we explored the effects of interventions with the trypanosomicidal drug Benznidazole (Bz) alone or combined with Pentoxifylline (PTX), a methylxanthine derivative shown to modulate immunological and cardiac abnormalities in a model of chronic chagasic cardiomyopathy, on parasite load and expression of miR-145-5p and miR-146b-5p. The infection of H9C2 cells with trypomastigote forms allowed parasite cycle with intracellular forms multiplication and trypomastigote release. After 48 and 144 h of infection, upregulation of miR-145-5p (24 h: 2.38 ± 0.26; 48 h: 3.15 ± 0.9-fold change) and miR-146b-5b (24 h: 2.60 ± 0.46; 48 h: 2.97 ± 0.23-fold change) was detected. The peak of both miRNA levels paralleled with release of trypomastigote forms. Addition of 3 µM and 10 µM of Bz 48 h after infection reduced parasite load but did not interfere with miR-145-5p and miR-146b-5p levels. Addition of PTX did not interfere with Bz-induced parasite control efficacy. Conversely, combined Bz + PTX treatment decreased the levels of both microRNAs, resembling the expression levels detected in non-infected H9C2 cells. Moreover, the use of miR-145-5p and miR-146b-5p mimic/inhibitor systems before infection of H9C2 cells decreased parasite load, 72 h postinfection. When H9C2 cells were treated with miR-145-5p and miR-146b-5p mimic/inhibitor 48 h after infection, all the used systems, except the miR-146b-5p inhibitor, reduced parasite load. Altogether, our data indicate that these microRNAs putatively control signaling pathways crucial for parasite–host cell interaction. Thus, miR-145-5p and miR-146b-5p deserve to be further investigated as biomarkers of parasite control and tools to identify therapeutic adjuvants to etiological treatment in Chagas disease.

www.nature.com/scientificreports/ of these miRNA as biomarkers of parasite control, evaluating their expression levels under treatment of infected cells with the trypanosomicidal drug Benznidazole (Bz) alone or combined with the immunoregulator Pentoxifylline (PTX).

Materials and methods
Cell culture. H9C2 (2-1) rat cardiomyoblast cell line was purchased from Banco de Células do Rio de Janeiro (BCRJ, code: 0098) and cultured in high glucose (4500 mg/L) Dulbecco's Modified Eagle's medium (DMEM) supplemented with pyrogen-free 10% fetal bovine serum (FBS) and 100 μg/mL penicillin/streptomycin under an atmosphere of 5% CO 2 at 37 °C. VERO cells were cultured in RPMI medium supplemented with HEPES, 5% FBS and 100 μg/mL penicillin/streptomycin under an atmosphere of 5% CO 2 at 37 °C. Both cell lines were submitted to Mycoplasma detection following a previously stablished protocol 52,53 . All reagents were purchased from ThermoFisher Scientific, Brazil. Cellular viability assay. Cellular viability was verified in 1 × 10 3 cells previously grown in a 96-well plate under an atmosphere of 5% CO 2 at 37 °C. Cells were treated with DMSO at 0.1%, Bz, PTX and the combined therapy of Bz + PTX. Cells were grown under treatment for 96 h under an atmosphere of 5% CO 2 at 37 °C and cellular viability was determined using Thiazolyl Blue Tetrazolium Bromide (MTT) assay following protocol previously described 55 . The plates were read at 570 nm using a Molecular Devices SpectraMax Plus microplate reader (California, USA). The experiment was repeated three times, using different cell culture flasks in each experimental condition.

H9C2 cells infection.
Transfection with miR-145-5p and miR-146b-5p mimic/inhibitor system. MicroRNAs mir-Vana miRNA mimic for miR-145-5p (ThermoFisher, USA. Assay ID MC11480) and miR-146b-5p (Assay ID MC10105) and inhibitor systems for miR-145-5p (Assay ID MH11480) and miR-146b-5p (Assay ID MH10105) were used according to manufacturer's instructions using Lipofectamine RNAiMAX reagent (Invitrogen, Ther-moFisher, USA) and Opti-MEM medium. Mimic and inhibitor systems for miR-145-5p and mimic system for miR-146b-5p were used at the final concentration of 10 pmol and inhibitor system for miR-146b-5p was used at the final concentration of 30 pmol in 6-well plates with previously added 5 × 10 4 H9C2 cells and incubated under an atmosphere of 5% CO 2 at 37 °C. All controls are added with Lipofectamine RNAiMAX. The experiment was repeated three times, using different cell culture flasks in each experimental condition.   1D). The number of intracellular forms per each cell is easily visualized in the microscopy images, as we detected a proportional amount of amastigote forms inside the cardiomyoblasts as MOIs were increased, at 24 and 48 hpi (Fig. 1E). These results show that H9C2 is a suitable host cell-T. cruzi interaction, and to that purpose we have chosen the MOI of 10:1 to proceed with the subsequent experiments. Thus, we evaluated the capability of T. cruzi to complete its lifecycle within the H9C2 cell line. For that, we infected H9C2 cells with 10:1 T. cruzi trypomastigotes for 4 h to allow parasite-cell interaction and collected samples at 4, 6, 8, 10, 24, 48 and 144 hpi to assess parasite load, released trypomastigotes at supernatant and levels of miR-145-5p and miR-146b-5p levels ( Fig. 2A). The kinetic of parasite load inside cells was evaluated at different timepoints, and although parasite load (parasite equivalents) could be detected at 4, 6, 8, 10, 24 and 48 hpi, being significantly increased at 10 hpi and 144 hpi (1413.0 ± 1162.0), compared to the 8 hpi (Fig. 2B). This result shows that at least at 10 hpi the intracellular parasites have not yet undergone into an intense auto-replicative process, which spiked after 144 hpi. The photo documentation of the kinetic of infection shows single intracellular forms at 4, 8, 10, 24 hpi, while amastigote nests were seen at 48 hpi, with significant increase at 144 hpi, when amastigote and trypomastigote forms coexist inside large nests (Fig. S2). Extracellular T. cruzi trypomastigote forms were only detected at 144 hpi (1.41 ± 0.30 × 10 6 trypomastigotes/mL) (Fig. 2C). Therefore, H9C2 cells allowed the complete life cycle of the Colombian T. cruzi strain, as described for mammal cells 60 , and showed to be a useful model for in vitro experiments to challenge our questions.  www.nature.com/scientificreports/ (D) miR-145-5p levels, (E) miR-146b-5p levels. For all graphs, significance was determined using unpaired Student's t test (*p < 0.05, **p < 0.01, ***p < 0.001). The experiment was repeated three times, using different cell culture flasks in each experimental condition. www.nature.com/scientificreports/ and 24 hpi but showed significant augmentation at 48 (2.60 ± 0.46) and 144 (2.97 ± 0.23) hpi (Fig. 2E). Thus, these data revealed that the upregulation of expression of the miR-145-5p and miR-146b-5p precedes the release of the trypomastigote forms of the parasite from the H9C2 cells, and the highest levels of both microRNAs paralleled the release of the trypomastigote forms. In order to confirm the specificity of the miR-145-5p and miR-146b-5p TaqMan assays to the miRNAs from H9C2 cells and the absence of cross-reaction with T. cruzi total RNA, 10 ng of total RNA, extracted from T. cruzi trypomastigotes, was reversed transcribed using the miR-145-5p and miR-146b-5p stem-loop primers, and real time PCRs targeting miR-145-5p and miR-146b-5p were carried out using 2 µL T. cruzi cDNA, as described in "Materials and methods". As reported in Fig. S3, no cross-amplification of T. cruzi RNA was detected in the assays, confirming the specificity of the TaqMan assays used herein.
Positive and negative modulation of miR-145-5p and miR-146b-5p in H9C2 cells affects T. cruzi replication. After observing the upregulation of miR-145-5p and miR-146b-5p levels in T. cruzi-infected H9C2 cells, we evaluated the effects of up-and down-regulation of these miRNAs in the in vitro experimental infection using a TaqMan miRNA mimic/inhibitor transfection system. First, we assessed the transfection protocol effect on cell viability adding 5, 10 and 30 pmol of miR-145-5p and miR-146b-5p mimic/inhibitor system and collecting cells 96 h hours later. For miR-145-5p, only at concentrations of 10 (84.32 ± 2.39% of viable cells) and 30 pmol (83.68 ± 9.99% of viable cells) of the mimic system a decreasing in cell viability was observed, compared to the control (Fig. S5A). Regarding miR-146b-5p, none of the concentrations used for both mimic and inhibitor systems were capable of decreasing cell viability (Fig. S5B). Therefore, the concentration of 5 pmol was chosen for subsequent experiments, except for the inhibitor system of miR-146b-5p, where the concentration of 30 pmol was chosen, once 5 pmol and 10 pmol was not effective in decreasing gene expression, as this microRNA already has a low basal level in H9C2 cells. Next, H9C2 cells were transfected with miR-145-5p and miR-146b-5p mimic/inhibitor systems (Fig. S5C). miR-145-5p mimic system successfully increased its expres- www.nature.com/scientificreports/ www.nature.com/scientificreports/ sion at both 24 (16,661 ± 1166 times higher than non-transfected cells) and 48 h (34,182 ± 3910 times higher than non-transfected cells) compared to control (Fig. S5D). Additionally, miR-145-5p inhibitor system also showed efficient transfection decreasing gene expression at 24 (0.14 ± 0.02 times lower than non-transfected cells) and 48 (0.20 ± 0.04 times lower than non-transfected cells) hours, compared to controls (Fig. S5E). miR-146b-5p mimic system worked efficiently at 24 (166.60 ± 11.66 times higher than non-transfected cells) and 48 h (341.80 ± 39.10 times higher than non-transfected cells) compared to the control (Fig S5F), and the inhibitor system also promoted decreasing in gene expression at 24 h (0.51 ± 0.13 times lower than non-transfected cells) compared to controls (Fig. S5G). Subsequently, we evaluated the effect of transfecting cells prior to infection in parasite load according to Fig. 4A. MicroRNA miR-145-5p modulation resulted in a decrease in parasite load in both mimic (755.70 parasite equivalents ± 282.20) and inhibitor (850.30 parasite equivalents ± 384.50) systems compared to control (1461 parasite equivalents ± 380.90) (Fig. 4B). Modulation of miR-146b-5p also resulted in a decrease in parasite load in both mimic (417.20 ± 58.12 parasite equivalents) and inhibitor (489.80 ± 117.50 parasite equivalents) systems compared to control (1461 ± 380.90 parasite equivalents) (Fig. 4C). Next, we evaluated the effects of transfecting cells after T. cruzi infection according to Fig. 4D. MicroRNA miR-145-5p modulation resulted in a decrease in parasite load in both mimic (824.70 ± 217.20 parasite equivalents) and inhibitor (813.30 ± 386.60 parasite equivalents) systems compared to controls (1743 ± 360.30 parasite equivalents) (Fig. 4E), and miR-146b-5p modulation also resulted in a significant decrease in parasite load in mimic (1032 ± 422.6 parasite equivalents) but not inhibitor (1515 ± 548.50 parasite equivalents) systems compared to controls (2067 ± 352.20 parasite equivalents) (Fig. 4F). These results come as an interesting finding, showing that positive and negative modulation of microRNAs prior and after T. cruzi infection may alter the parasite cycle dynamics within the cell, promoting decreasing in parasite load.

Discussion
Chronic chagasic cardiomyopathy is the most frequent and severe form of CD, being the main cause of morbidity by cardiovascular impairment in endemic areas 63 . Efforts have been made to understand the role of global alterations of microRNAs expression profiles in controlling CCC pathogenesis related pathways 33,34 and previous studies focused on specific microRNA profiles 32,64 . The cardiac tissue is an important site for T. cruzi persistence in the host during the chronic phase of infection 65 , therefore, in this study, firstly we propose the validation of an in vitro experimental model to investigate T. cruzi interactions with the host cell using the rat H9C2 cardiomyoblast cell line, which will spare the use of animals to obtain primary cardiomyocyte cultures and speed off the investigation of parasite-host interactions. The Colombian T. cruzi strain infection of H9C2 cells revealed increased intracellular parasite load and release of trypomastigote forms at 144 hpi, suggesting a successful infection and the complete parasite cycle, as expected to be detected in a mammal cell 60 . Although the Colombian T. cruzi strain is knowingly recognized as cardiotropic, it was also able to successfully infect and complete the parasite cycle in astrocytes, the main parasite auberge in the central nervous system 66,67 . Altogether, our results suggest that the H9C2 cardiomyocyte cell line is capable of sustaining the lifecycle of the Colombian T. cruzi strain and, therefore, is a suitable cell for in vitro experimental model of CD to explore host-parasite interaction. Additionally, primary cardiomyocytes and H9C2 show similar hypertrophic responses, and might be interchangeable for prospective molecular studies in heart development and diseases 68 . Amastigote forms of T. cruzi started intracellular development after 24 hpi and showed increase of numbers at 48 hpi, meaning they are going through intense division, suggesting that T. cruzi replication and releasing might be causing, directly or indirectly, upregulation of these microRNAs. It is well known that during the T. cruzi-cardiomyocyte interaction, the parasite start controlling overall host gene expression, including immune response genes, inflammation, cytoskeletal organization among other features 69 , therefore, it is most likely that T. cruzi also takes over microRNAs expression. Potential targets for the miR-145 family present in cardiovascular cells include SMAD3, a member of the SMAD family that acts as mediator of the signaling pathway triggered by TGF-β, and PAK1 and PAK4, molecules that play a role in cytoskeleton remodeling, affecting different cellular processes as directional motility, invasion, metastasis, and growth 39 . Thus, the upregulated miR-145-5p might act downregulating genes related to cell organization and growth processes affected by T. cruzi infection. A notable result is the increasing levels of miR-145-5p and miR-146b-5p in infected H9C2 cells at 48 hpi, observed before trypomastigotes releasing at 144 hpi. These findings led us to propose that this event should be explored in an in vivo model of chronic CD reactivation. If it is shown to be true, the use of the miR-145-5p and miR-146b-5p miRNAs as surrogate biomarkers could be potentially useful in patients subjected to immunosuppression after heart transplantation, as cardiac biopsies are taken in regular intervals to assess reinfection and could be an important tool to detect those miRNAs even before the increasing of the parasite load in the heart and blood 70 , allowing health professionals to make proper interventions in time to prevent reactivation and its consequences.
The infection caused by T. cruzi elicits an immune response that is driven by pro-inflammatory cytokines such as IFN-γ and TNF, chemokines and enzymes and has been shown in several studies that etiological treatment contributes to parasite load reduction and rearrangement of the dysregulated immune response in patients 71,72 and experimental models 15,71 . Chagas disease cardiomyopathy is a knowingly immune dysregulated disorder 46 , and recent study by our group, using C57BL/6 mice chronically infected with the same Colombian T. cruzi strain used in the present study, showed global immune dysregulation with upregulation of key CD cytokines such as IFN-γ, CSF2, IL-12, IL-2 and chemokines such as CCR4, CCL3 and CCL5 73 corroborating that infection with T. cruzi is in fact causing increased production of inflammatory mediators that could be triggering the upregulation of miR-145-5p and, especially miR-146b-5p that is highly dependent of inflammatory stimulus 44 . Besides its trypanosomicidal activity, Bz has also been explored as having anti-inflammatory properties 14 . In a model of primary cardiomyocyte culture infected with the RA T. cruzi strain, treatment with suboptimal doses has shown to decrease NOS2 expression, IL-1β and IL-6 production 11 . Additionally, recent study from our group on a 30-day www.nature.com/scientificreports/ www.nature.com/scientificreports/ treatment with Bz in the C57BL/6 T. cruzi chronic model, showed downregulation of altered pro-inflammatory molecules as CSF2, IL-7 and IL-12, also substantially decreasing the production of IFN-γ and IL-2 73 , which was not sufficient however, to reverse the upregulation of both miRNAs triggered by the infection, although it was effective in controlling parasite replication. A previous study of microRNA transcriptome profiling found miR-146b-5p to be upregulated and associated with parasitemia levels and electrical abnormalities in a model of acute Chagas' heart disease induced by infection with the Colombian strain 33 , corroborating our data and supporting the importance of therapeutic intervention aiming to decrease the expression of this miRNA. Pentoxifylline is a hemorheological agent that has described anti-inflammatory and antioxidant effects 16 , however, it has no trypanosomicidal effect, meaning that the failure to decrease parasite load showed here was expected and previously described in in vivo model of infection 19 . Moreover, PTX has been studied as an immunomodulatory agent complementary to etiological treatment in CD 15 and in other parasitic infectious diseases such as cutaneous 74,75 and mucosal 76,77 leishmaniasis. Nevertheless, the combined therapy was effective in reversing the upregulation of both miR-145-5p and miR-146b-5p. Additionally to its classically described mechanism of action, PTX can also inhibit a wide range of cytotoxic responses mainly by acting on cytokine production dynamics 78 , downregulating proinflammatory cytokines IFN-γ, GM-CSF and TNF 79 , attenuating cell surface expression of IL-2 receptor and production of IL-8 and CCL2 in pulmonary epithelial cells 80 . Recent study from our group on a 30-day treatment with Bz + PTX in the C57BL/6 T. cruzi chronic model, showed regulation of upregulated pro-inflammatory molecules as CSF2, IL-7 and IL-12, also substantially decreasing the production of IFN-γ, CSF2, IL-2 among other immunological molecules 73 suggesting that the immunomodulation caused by the combined Bz + PTX therapy might be affecting the upregulation of these two miRNAs. It is most likely that the combined Bz + PTX therapy was able to reverse miR-145-5p and miR-146-5p overexpression by combining the trypanosomicidal effect of Bz, successfully eliminating T. cruzi and, putatively, reducing the tissue damage caused by the parasites, as well as the immunomodulatory effect of PTX, preventing potential overproduction of cytokines that could culminate in the modulation of potential miRNA targets, that remains to be explored in the present experimental T. cruzi model. Previously, miR-145 has been explored in cancer research, and cardiac system, mainly focusing on myocardial infarction [81][82][83][84][85][86][87] . Study using an in vitro model of hypoxia in H9C2 cells, showed upregulation of miR-145 associated with decrease in cell viability and apoptosis, revealing Rac1 (Rac family small GTPase 1) as a target and showing the involvement of PI3K/Akt and MAPK/ERK signaling pathways 82 , which was further confirmed by in vivo experiments 86 . In T. cruzi, actin-regulating molecules such as Rac1 CDc42 have been implicated in amastigote invasion, mediating actin recruitment and enhancing invasiveness 88 . It is known that miR-146b-5p is dependent on inflammatory stimulus of specific cytokines such as IFN-γ, TNF and IL-1β 44 , that are also dysregulated in CD 5 , as showed in recent study by our group in cardiac tissue using an experimental model of C57BL/6 mice chronically infected with the Colombian T. cruzi strain 73 . In murine T. cruzi infection, PTX was able to reverse CCC clinical signs, hampering the progression of heart injury, as improves connexin 43 expression and decreases fibronectin overdeposition, reducing CD8 + T-cells expressing activation and migration markers, and of activated blood vessel endothelial cells, and decreased the number of perforin-expressing cells invading the cardiac tissue 19 . Recently, Bz was shown to reduce of IL-6 and TNF production by T. cruzi-infected cardiac spheroids 12 and promote down-regulation of NF-kB by LPS-stimulated cardiomyocytes 11 . Therefore, all these results reinforce the importance of proposing a combined etiological and immunomodulatory treatment for CCC.
The up and downmodulation of miR-145-5p and miR-146b-5p using TaqMan mimic/inhibitor systems as a pre-treatment before T. cruzi infection and as a post-treatment after T. cruzi infection to evaluate if the miRNA modulation would affect parasite load in H9C2 cardiomyoblast cells. Study on T. cruzi infection of Schwann cells revealed that trans-sialidase triggers the survival of host cell via the PI3K/Akt pathway through activation of PI3K, helping the establishment of a successful infection stimulating host antiapoptotic mechanisms 89 . Additionally, a more recent study showed that extracellular amastigotes of T. cruzi can activate Akt and ERK molecules, suggesting the participation of both PI3K/Akt and MAPK/ERK signaling pathways, interfering with cytoskeleton rearrangement and phagocytosis, probably subverting the phagocytic machinery allowing a successful infection 90 . It is also known that miR-145-5p specifically affects these pathways 82 . In T. cruzi, Rac1 has been previously elucidated as the molecule modulating actin recruitment and enhancing amastigote invasiveness 88,91 . Moreover, Rac1 is a known target of miR-145-5p 82 , thus it is possible that pre-and pos-treatment of cells with miR-145-5p mimic system might be downregulating molecules of this pathway, therefore hampering parasite-host interaction dynamics and impairing the establishment of a successful infection. However, the role played by pre-and post-treatment with miR-145-5p inhibitor system also promoting decrease in parasite load deserves further investigation, once it might be activating signaling pathways that are yet unknown, providing disadvantage to the parasite in the infection. Moreover, pre-and post-treatment with miR-146b-5p mimic/inhibitor systems was also able to significantly decrease infection, except from the post-treatment inhibitor system, which silencing through TaqMan inhibitor system was not as effective in silencing the expression of miR-146b-5p, suggesting the lack of efficiency in reducing parasite load. Treatment with miR-146b-5p mimic might contribute to the downregulation of known targets such as IRAK1 and TRAF6, involved in the TLR4 signaling pathway, and TIMP-4, MMP16 and TGIF1 48,49 . However, pre-treatment with miR-146b-5p inhibitor system seems more prominent once the downregulation of this microRNA might cause the upregulation of these targets. It has been recently discovered that the use of a TLR4 agonist promotes higher survival rate and decreases parasite burdens in BALB/c mice acutely infected with T. cruzi, although cardiac damage was not prevented 92 , thus shedding light on how miR-146b-5p is hampering infection of cells and decreasing parasite load. Additionally, silencing of miR-146b-5p in a murine and porcine model of myocardial infarction reduced fibrosis and cell death, restoring cardiac remodeling and heart function 51 , which shows us additional advantages to the further investigation of the use of miR-146b-5p for the treatment of CCC, as well as a tool for identification of putative pathways to be targeted by new therapeutic approaches. www.nature.com/scientificreports/ There are several other miRNAs involved in CD establishment and progression [31][32][33][34][35]37,64 . Nevertheless, we evaluated the modulation of two miRNAs based on their regulation in experimental acute CD in previous studies 34,93 , and the involvement of miR-145-5p in cardiomyopathies 82,83,86,[94][95][96] and miR-146b-5p in inflammatory processes 97,98 that are known to be dysregulated in CD. The evaluation of only two miRNAs and the absence of the expression analysis of other host mRNAs that can be related to the infection control are a limitation of this study. Additionally, the evaluation of specific time points in between 48 and 144 h in the time-course infection experiment are missing, once we thought it would be more relevant to evaluate the modulation of both miRNAs in early stages of the infection when T. cruzi trypomastigotes are in the process of entering the host cell, and in late time points when the infection is already established and trypomastigotes are being actively released from cardiomyocytes, crucial biological processes to support parasite persistence in the heart tissue 69,99,100 . Nevertheless, the results observed here are clear, opening a venue to the search of these miRNAs as new biomarker candidates to monitor the etiological treatment in CD, especially for prognosis prediction and therapeutic cure in the chronic phase of CD 101 . The gold standard assessment for treatment efficacy is still seroconversion of serological tests, which may take decades to become negative 72,101 . In this context, microRNAs could emerge as potential biomarkers mainly because they are present in the circulation and have stable detection, due to several mechanisms that prevent their degradation 102 . miR-1, for example, was firstly reported as a key regulator of cardiac differentiation 103 and was later validated as potential biomarker in heart failure onset after myocardial infarction, as it showed to negatively correlate with ejection fraction on 49 patients tested 104 . In Chagas disease, miR-208a, a heart specific miRNA that plays a critical role in cardiac dysfunction leading to heart failure, showed increased circulating levels during the chronic indeterminate phase, suggesting that this microRNA could be a potential risk-prediction score biomarker for CCC 36 . Another study investigated six prominent miRNAs involved on cardiac remodeling, cardiac hypertrophy and fibrosis, observed differential expression for miR-19a, miR-21-5p and miR-29b-3p in patients with CCC when compared to indeterminate form patients, that positively correlated with cardiac dysfunction and fibrosis and negatively correlated with ejection fraction, suggesting the potential of these microRNAs as biomarkers of CCC progression 37 . Since it is well known that one single miRNA may target many mRNA transcripts regulating several pathways depending on the condition 20,105 , the participation of miR-145-5p and miR-146b-5p in pathophysiological processes of T. cruzi infection might be more relevant than previously thought. It is clear from the results we came across in this study the participation of miR-145-5p and miR-146b-5p in the parasite-host interaction and establishment of T. cruzi infection. Additionally, pharmacotherapy being able to modulate these microRNAs might serve as a first insight on the potential role of microRNAs as biomarkers of treatment efficacy, although further validation is needed. Furthermore, the potential use of miR-145-5p and miR-146b-5p as therapeutic adjuvants in the treatment of CD also deserves special attention once they might be acting on the parasite entrance in host cells and could improve traditional Bz treatment efficacy.